Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Jong, Annika de; Dirven, Richard; Oud, Josine A.; Tio, David; Vlijmen, Bart J.M. van; Eikenboom, Jeroen

doi:10.1111/jth.14140

Cited by 19 publications

(35 citation statements)

References 38 publications

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“…For determination of intracellular VWF, cells were lysed overnight at 4°C in Opti‐MEM™ I Reduced Serum Medium (Thermo Fisher Scientific, Carlsbad, CA) containing 0.1% Triton X‐100 (Sigma‐Aldrich) supplemented with cOmplete™ Protease Inhibitor Cocktail with EDTA (Roche Diagnostics, Mannheim, Germany). VWF:Ag levels were measured in conditioned medium and cell lysates by ELISA as previously described …”

Section: Methodsmentioning

confidence: 99%

Variability of von Willebrand factor‐related parameters in endothelial colony forming cells

Jong

Weijers

Dirven

et al. 2019

Journal of Thrombosis and Haemostasis

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Essentials Endothelial colony forming cells (ECFCs) are a powerful tool to study vascular diseases ex vivo.Separate ECFC lines show variations in morphology and von Willebrand factor‐related parameters.Maximum cell density is correlated with von Willebrand factor expression in ECFCs.Variations in ECFC lines are dependent on the age and mesenchymal state of the cells. AbstractBackgroundEndothelial colony forming cells (ECFCs) are cultured endothelial cells derived from peripheral blood. ECFCs are a powerful tool to study pathophysiological mechanisms underlying vascular diseases, including von Willebrand disease. In prior research, however, large variations between ECFC lines were observed in, among others, von Willebrand factor (VWF) expression.ObjectiveUnderstand the relation between phenotypic characteristics and VWF‐related parameters of healthy control ECFCs.MethodsECFC lines (n = 16) derived from six donors were studied at maximum cell density. Secreted and intracellular VWF antigen were measured by ELISA. The angiogenic capacity of ECFCs was investigated by the Matrigel tube formation assay. Differences in expression of genes involved in angiogenesis, aging, and endothelial to mesenchymal transition (EndoMT) were measured by quantitative PCR.ResultsDifferent ECFC lines show variable morphologies and cell density at maximum confluency and cell lines with a low maximum cell density show a mixed and more mesenchymal phenotype. We identified a significant positive correlation between maximum cell density and VWF production, both at protein and mRNA level. Also, significant correlations were observed between maximum cell density and several angiogenic, aging and EndoMT parameters.ConclusionsWe observed variations in morphology, maximum cell density, VWF production, and angiogenic potential between healthy control ECFCs. These variations seem to be attributable to differences in aging and EndoMT. Because variations correlate with cell density, we believe that ECFCs maintain a powerful tool to study vascular diseases. It is however important to compare cell lines with the same characteristics and perform experiments at maximum cell density.

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Section: Methodsmentioning

confidence: 99%

Variability of von Willebrand factor‐related parameters in endothelial colony forming cells

Jong

Weijers

Dirven

et al. 2019

Journal of Thrombosis and Haemostasis

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“…VWF multimers were visualized using agarose gel electrophoresis under nonreducing conditions as described before. 17 VWF multimers were visualized using ECL Western Blotting Substrate (Promega, Madison, Wisconsin, United States).…”

Section: Vwf Protein Analysismentioning

confidence: 99%

“…This is in line with results in HEK293 cells. 17 Correction of a VWD Type 2A Multimerization Phenotype by an Allele-Specific siRNA…”

Section: Allele-specific Sirna Inhibition In Healthy Control Ecfcsmentioning

confidence: 99%

“…We recently published the proof of principle of this approach in human embryonic kidney (HEK)293 cells overexpressing VWF alleles. 17 We showed that allele-specific siRNAs that target a heterozygous single-nucleotide polymorphism (SNP) located on the same allele as a dominant-negative VWD type 2A mutation corrects for the VWD phenotype. 17 For this approach, various siRNAs have been selected to target four frequent SNPs in VWF.…”

Section: Introductionmentioning

confidence: 99%

“…17 We showed that allele-specific siRNAs that target a heterozygous single-nucleotide polymorphism (SNP) located on the same allele as a dominant-negative VWD type 2A mutation corrects for the VWD phenotype. 17 For this approach, various siRNAs have been selected to target four frequent SNPs in VWF. It was calculated that 74% of the Caucasian population is heterozygous for at least one of these four SNPs and thus might be a candidate for this approach of allele-specific VWF silencing.…”

Section: Introductionmentioning

confidence: 99%

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Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA

et al. 2020

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Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

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ADAMTS‐13 and bleeding phenotype in von Willebrand disease

Boender

Nederlof

Meijer

et al. 2020

Research and Practice in Thrombosis and Haemostasis

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Background The bleeding phenotype of von Willebrand disease (VWD) varies highly between patients and can only partly be explained by von Willebrand factor (VWF) parameters. By cleaving large VWF multimers into smaller, less active multimers, ADAMTS‐13 is an important regulator of VWF activity. However, it is unknown what the role of ADAMTS‐13 is in individuals with VWD. Objectives We therefore studied how ADAMTS‐13 activity is associated with the laboratory and bleeding phenotype in individuals with VWD. Methods We measured ADAMTS‐13 activity using the fluorescence resonance energy transfer substrate VWF 73 assay in 638 individuals with VWD in the nationwide cross‐sectional Willebrand in the Netherlands study and in 36 healthy controls. The bleeding phenotype was assessed using the Tosetto bleeding score. Results ADAMTS‐13 activity was similar in individuals with VWD (109% ± 20.6%) and controls (110% ± 19.7%). ADAMTS‐13 activity was higher in individuals with VWD with type 3 than those with type 1 (mean difference, 11.8%; 95% confidence interval [CI], 2.9%‐20.8%) or type 2 (mean difference, 16.1%; 95% CI, 7.1%‐25.1%). ADAMTS‐13 activity was not associated with the Tosetto bleeding score (0.1 Tosetto bleeding score increase per 10% ADAMTS‐13 increase, 95% CI, −0.2 to 0.3). Furthermore, ADAMTS‐13 activity did not differ between individuals with and without a bleeding event during the year preceding blood sampling (mean difference, 1.4%; 95% CI, −2.1% to 4.9%). Conclusion ADAMTS‐13 activity was highest in individuals with type 3 VWD, but it had only minor associations with VWF parameters. ADAMTS‐13 activity does not influence the bleeding phenotype in individuals with VWD.

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Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Cited by 19 publications

References 38 publications

Variability of von Willebrand factor‐related parameters in endothelial colony forming cells

Variability of von Willebrand factor‐related parameters in endothelial colony forming cells

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA

ADAMTS‐13 and bleeding phenotype in von Willebrand disease

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